Contents lists available at ScienceDirect Bone journal homepage: www.elsevier.com/locate/bone Full Length Article Mutations in the Neuroblastoma Amplified Sequence gene in a family affected by Acrofrontofacionasal Dysostosis type 1 Eleonora Palagano a,b,1 , Giulia Zuccarini c,1 , Paolo Prontera d , Renato Borgatti e , Gabriela Stangoni d , Sandro Elisei f , Stefano Mantero a,g , Ciro Menale a,g , Antonella Forlino h , Paolo Uva i , Manuela Oppo i , Paolo Vezzoni a,g , Anna Villa a,g , Giorgio R. Merlo c , Cristina Sobacchi a,g, ⁎ a Humanitas Clinical and Research Institute, via Manzoni 113, 20089 Rozzano, Italy b Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Vanvitelli 32, 20133 Milan, Italy c Department Molecular Biotechnology and Health Sciences, University of Turin, Via Nizza 52, 10126 Turin, Italy d Centro di Riferimento Regionale di Genetica Medica, Azienda Ospedaliera di Perugia, Piazzale Menghini 8/9, 06129 Perugia, Italy e Child Neuropsychiatry and Neurorehabilitation Department, Scientific Institute Eugenio Medea, La Nostra Famiglia, Via Don Luigi Monza 20, 23842 Bosisio Parini, Italy f Istituto Serafico di Assisi, Viale Guglielmo Marconi 6, 06081 Assisi, Italy g CNR-IRGB, Milan Unit, via Fantoli 16/15, 20138 Milan, Italy h Department of Molecular Medicine, Unit of Biochemistry, University of Pavia, Via Taramelli 3/B, 27100 Pavia, Italy i CRS4, Science and Technology Park Polaris, Loc. Piscina Manna, 09010 Pula, Italy ARTICLE INFO Keywords: Dysostosis NBAS Nonsense-mediated decay Retrograde transport Development ABSTRACT Acrofrontofacionasal Dysostosis type 1 (AFFND1) is an extremely rare, autosomal recessive syndrome, com- prising facial and skeletal abnormalities, short stature and intellectual disability. We analyzed an Indian family with two affected siblings by exome sequencing and identified a novel homozygous truncating mutation in the Neuroblastoma-Amplified Sequence (NBAS) gene in the patients' genome. Mutations in the NBAS gene have recently been associated with different phenotypes mainly involving skeletal formation, liver and cognitive development. The NBAS protein has been implicated in two key cellular processes, namely the non-sense mediated decay and the Golgi-to-Endoplasmic Reticulum retrograde traffic. Both functions were impaired in HEK293T cells overexpressing the truncated NBAS protein, as assessed by Real-Time PCR, Western blot analysis, co-immunoprecipitation, and immunofluorescence analysis. We examined the expression of NBAS protein in mouse embryos at various developmental stages by immunohistochemistry, and detected expression in devel- oping chondrogenic and osteogenic structures of the skeleton as well as in the cortex, hippocampus and cere- bellum, which is compatible with a role in bone and brain development. Functional genetics in the zebrafish model showed that depletion of endogenous z-nbas in fish embryos results in defective morphogenesis of chondrogenic cranial skeletal elements. Overall, our data point to a conserved function of NBAS in skeletal morphogenesis during development, support the hypothesis of a causative role of the mutated NBAS gene in the pathogenesis of AFFND1 and extend the spectrum of phenotypes associated with defects in this gene. 1. Introduction In mammals the axial skeleton, the limb skeleton and the cranio- facial bones derive from three distinct embryonic structures through two different mechanisms: endochondral and intramembranous ossifi- cation. In the former, typical of the long bones and axial skeleton, a cartilaginous template is generated from the condensation of me- senchymal cells and then remodeled into mature bone; in the latter, giving rise to flat bones, osteoblast progenitors directly differentiate and mature from the condensed mesenchyme. A number of molecules, comprising transcription factors and growth factors, carry out pivotal roles; however, the spatial and temporal regulation of skeletogenesis is not yet fully unraveled [1]. Genetic alterations of these processes are responsible for a huge variety of disorders affecting the skeletal system. In particular, dysos- toses (ORPHA: 364559) arise from defects in embryonic development; different skeletal compartments can be affected and symptoms at other organs can be present, leading to a high heterogeneity of https://doi.org/10.1016/j.bone.2018.06.013 Received 25 January 2018; Received in revised form 14 June 2018; Accepted 17 June 2018 ⁎ Corresponding author at: Humanitas Clinical and Research Institute, via Manzoni 113, 20089 Rozzano, Italy. 1 These authors contributed equally to this work. E-mail address: cristina.sobacchi@humanitasresearch.it (C. Sobacchi). Bone 114 (2018) 125–136 Available online 19 June 2018 8756-3282/ © 2018 Elsevier Inc. All rights reserved. T